This invention relates to a method of quantitative analysis of cyclic nucleotides depending on the enzyme immunoassay method (hereinafter referred to by the abbreviation EIA) and to reagents therefor.
In recent years, adenosine-3',5'-cyclic monophosphate (hereinafter referred to by the abbreviation cAMP) has been widely studied as a mediator of hormone action, and its physiologic actions with respect to guanosine-3',5'-cyclic monophosphate (cGMP), inosine-3',5'-cyclic monophosphate (cIMP), gytidine-3',5'-cyclic monophosphate (cCMP), and uridine-3',5'-cyclic monophosphate (cUMP) (hereinafter referred to respectively by the indicated abbreviations) are being clarified.
Particularly in the case where a living body assumes an unphysiologic or pathologic state such as that due to a disease, the contents of cAMP and cGMP in the cells or body fluids of that living body fluctuate. From this observation, the measurement of these contents within living bodies is being increasingly regarded to be of important significance not only in the basic medical research field but also for diagnosis, prevention, and treatment of diseases in the field of clinical medicine. For example, the determination of the contents of cAMP and cGMP in living body samples such as leucocytes of asthmatic patients, skins of psoriasis patients, blood platelets of thrombocytosis patients, blood and urine of psuedohypoparathyroidism patients, and cerebrospinal fluid of manic-depressive psychotic patients is considered to be effective in the diagnosis and treatment of these diseases. Furthermore, it is predictable that the relationships between these cyclic nucleotides and a large number of disease conditions will become clear in the future. Accordingly, there is a deep significance in the establishment of simple, convenient, and highly accurate methods of quantitatively determining cyclic nucleotides.
One of the methods of quantitative analysis of cyclic nucleotides such as cAMP and cGMP known heretofore is the so-called radioimmunoassay method (hereinafter referred to by the abbreviation RIA method). This RIA method comprises causing a cyclic nucleotide in a living body sample and a cyclic nucleotide labeled with a radioisotope to react competitively with a corresponding antibody and measuring the quantity of radiation of the radioisotope-labeled cyclic nucleotide which has bonded with the antibody or the radioisotope-labeled cyclic nucleotide which has not bonded with the antibody thereby to determine the quantity of the cyclic nucleotide.
The quantitative analysis with very high accuracy of cyclic nucleotides by applying to this RIA method such methods as the method in which a cyclic nucleotide is succinylated thereby to improve its affinity for an antibody obtained when cyclic nucleotide-succinyl albumin is used as an antigen and the method of using an imidazole buffer solution for the reaction medium of an antigen-antibody reaction thereby to increase the bonding rate, the sensitivity, and the stability of the bonded product is being reduced to practice, for example, as disclosed in Biochemical Medicine, Vol. 18, pp. 257-273, (1977).
These RIA methods, however, are accompanied by various problems basically arising from the use of radioisotopes. For example, there is the danger of harm to the human body and environmental pollution due to radiation. Another problem is the need for special facilities and equipment for preventing the diffusion of radiation. Still other problems are the need for expensive measuring apparatus for measuring radiation, the requirement for a qualification for handling radioisotopes, and the inconvenience of handling, transportation, and preservation of radioisotopes due to their instability, in general.
Recently, with the aim of solving these problems accompanying the RIA method, EIA methods in which enzymes are used as labels instead of radioisotopes have been studied and developed. Some are known to have already been reduced to practice as quantitative analysis methods for .alpha.-fetoprotein, HBs antigen, carcinoembryonic antigen, thyroxine, digoxin, IgE, etc. However, with regard to quantitative analysis by the EIA method of cyclic nucleotides, there has been no report whatsoever.
The present inventor has carried out research with the object of developing a method depending on the EIA method for quantitative analyses of cyclic nucleotides. As a result, the inventor has succeeded in perfecting a method which, in sensitivity, accuracy, and reproducibility is comparable to or superior to the RIA method.